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- EMDB-52723: Human microtubule triplet in centrosomes of HeLa cells -

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Basic information

Entry
Database: EMDB / ID: EMD-52723
TitleHuman microtubule triplet in centrosomes of HeLa cells
Map dataSubtomogram average of microtubule triplet in human centrosomes of HeLa cells (consensus refinement)
Sample
  • Cell: Microtubule triplets in human centrosomes of HeLa cells
Keywordscentrosome / microtubule nucleation / microtubule triplet / centriole / cell cycle / cytoskeleton / tubulin
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 30.5 Å
AuthorsHofer FW / Pfeffer S
Funding support Germany, 4 items
OrganizationGrant numberCountry
German Research Foundation (DFG)DFG Schi 295/4-4 Germany
German Research Foundation (DFG)DFG SCHI 295/11-1 Germany
German Research Foundation (DFG)DFG PF 963/1-4 Germany
German Research Foundation (DFG)DFG PF 963/4-1 Germany
CitationJournal: Nat Methods / Year: 2023
Title: ELI trifocal microscope: a precise system to prepare target cryo-lamellae for in situ cryo-ET study.
Authors: Shuoguo Li / Ziyan Wang / Xing Jia / Tongxin Niu / Jianguo Zhang / Guoliang Yin / Xiaoyun Zhang / Yun Zhu / Gang Ji / Fei Sun /
Abstract: Cryo-electron tomography (cryo-ET) has become a powerful approach to study the high-resolution structure of cellular macromolecular machines in situ. However, the current correlative cryo- ...Cryo-electron tomography (cryo-ET) has become a powerful approach to study the high-resolution structure of cellular macromolecular machines in situ. However, the current correlative cryo-fluorescence and electron microscopy lacks sufficient accuracy and efficiency to precisely prepare cryo-lamellae of target locations for subsequent cryo-ET. Here we describe a precise cryogenic fabrication system, ELI-TriScope, which sets electron (E), light (L) and ion (I) beams at the same focal point to achieve accurate and efficient preparation of a target cryo-lamella. ELI-TriScope uses a commercial dual-beam scanning electron microscope modified to incorporate a cryo-holder-based transfer system and embed an optical imaging system just underneath the vitrified specimen. Cryo-focused ion beam milling can be accurately navigated by monitoring the real-time fluorescence signal of the target molecule. Using ELI-TriScope, we prepared a batch of cryo-lamellae of HeLa cells targeting the centrosome with a success rate of ~91% and discovered new in situ structural features of the human centrosome by cryo-ET.
History
DepositionFeb 4, 2025-
Header (metadata) releaseMar 19, 2025-
Map releaseMar 19, 2025-
UpdateMar 26, 2025-
Current statusMar 26, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52723.png

Downloads & links

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Map

FileDownload / File: emd_52723.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of microtubule triplet in human centrosomes of HeLa cells (consensus refinement)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
8.59 Å/pix.
x 128 pix.
= 1099.93 Å		8.59 Å/pix.
x 128 pix.
= 1099.93 Å		8.59 Å/pix.
x 128 pix.
= 1099.93 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 8.5932 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.048
Minimum - Maximum-0.91579497 - 1.8266711
Average (Standard dev.)0.04379249 (±0.3422255)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 1099.9296 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Subtomogram average of proximal region microtubule triplets in...

Fileemd_52723_additional_1.map
AnnotationSubtomogram average of proximal region microtubule triplets in human centrosomes of HeLa cells (subclass)
Projections & Slices
AxesZYX

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Additional map: Subtomogram average of distal region microtubule triplets in...

Fileemd_52723_additional_2.map
AnnotationSubtomogram average of distal region microtubule triplets in human centrosomes of HeLa cells (subclass)
Projections & Slices
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Histogram (log scale)

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Additional map: Subtomogram average of central region microtubule triplets in...

Fileemd_52723_additional_3.map
AnnotationSubtomogram average of central region microtubule triplets in human centrosomes of HeLa cells (subclass)
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map for subtomogram average of microtubule triplet...

Fileemd_52723_half_map_1.map
AnnotationHalf map for subtomogram average of microtubule triplet in human centrosomes of HeLa cells (consensus refinement)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map for subtomogram average of microtubule triplet...

Fileemd_52723_half_map_2.map
AnnotationHalf map for subtomogram average of microtubule triplet in human centrosomes of HeLa cells (consensus refinement)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Microtubule triplets in human centrosomes of HeLa cells

EntireName: Microtubule triplets in human centrosomes of HeLa cells
Components
  • Cell: Microtubule triplets in human centrosomes of HeLa cells

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Supramolecule #1: Microtubule triplets in human centrosomes of HeLa cells

SupramoleculeName: Microtubule triplets in human centrosomes of HeLa cells
type: cell / ID: 1 / Parent: 0
Details: Obtained from EMPIARC-200003 dataset of human centrosomes
Source (natural)Organism: Homo sapiens (human) / Strain: HeLa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 3.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 9.0 µm / Nominal defocus min: 5.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 30.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number subtomograms used: 2040
ExtractionNumber tomograms: 12 / Number images used: 2040 / Software: (Name: PyTom (ver. 0.971), Warp (ver. 1.09))
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 3.1)
Details: Subclassification for microtubule triplet topologies
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)

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